In recent years, the possible regulation, in vivo, of cellular enzyme activity by thiol:disulfide interchange has received increasing attention as an important cellular process. It is well documented that thiol:disulfide exchange may affect such biological functions as protein synthesis, protein degradation, Ca2+ metabolism, cell cycle events and cytoskeleton regulation. In this proposal we plan to establish whether the thiol:protein disulfide transhydrogenase named thioltransferase (TT) by Mannervik, is identical to glutaredoxin, found in E. coli and calf thymus by Holmgren. Based on a newly developed procedure for the isolation of homogeneous pig liver thioltransferase in 32% yields, mg quantities of thioltranferase are now available to carry out primary amino acid sequence analysis for comparison with that of glutardoxin from calf thymus as reported by Holmgren. Other evidence for comparison of the two enzymes will be obtained by testing TT in the glutathione dependent assay for ribonucleotide reductase and by immunological studies. TT from pig liver has four cysteine groups. As a function of (H+), we plan to study the kinetics of reactivity of each cysteine to iodoacetic acid for analysis of their relative thiolate pKs. Preliminary results indicate that the cysteine closet to the N-terminus has an unusually low pK which may impart special properties to TT as a catalyst in the transfer of mixed disulfides. In addition, mechanistic studies of the transhydrogenase reaction between glutathione and liver proteins as catalyzed by TT will be undertaken. In the second section of this proposal, we plan to study potential functional roles for TT by investigating the activation/deactivation of key regulatory enzymes through thiol:protein disulfide interaction as catayzed by TT including phosphofructokinase and the eIF-2alpha kinase of the rabbit reticulocyte lysate protein synthesis system. In other studies, we wish to extend our preliminary observations into the TT catalyzed exchange between (35S)GSSG and rat liver proteins. Thus, the primary goals of this proposal are first to provide detailed structural and mechanistic information of pig liver TT that will eventually establish whether TT and glutaredoxin are identical proteins, and secondly, to provide preliminary information on the possible functional roles of TT as an essential thiol:protein disulfide carrier in specific cellular processes, in vitro.